The Study On Killing Spinal Astrocytes Using Upconversion Nanoparticles Mediated Photodynamic Therapy In Vitro

Objective:To investigate the phototoxicity effects of the nanocompound of upconversion fluorescent nanoparticles as a carrier of photosensitizer and merocyanine540(UCNPs-MC540) on rat spinal astrocytes in vitro.Methods:(1) Spinal cord dissected under sterile operation from rats aged1to2days were made to be cells suspension in the experiment. Differential attachment and orbital shaking bed were used to remove contaminating cells and the passaged cell was identified with immunofluorescence staining against glial fibrillary acidic protein (GFAP).(2) The spinal astrocytes cell were cultured successfully and then incubated with the UCNPs-MC540of various concentrations and exposured980nm infrared laser irradiation of different energy densities. The cell survival rates of each group were detected by MTT assay. Finally, morphology of astrocytes incubated with200μg/ml UCNPs-MC540after laser irradiation of2000J/cm2was observed via transmission electron microscope.Results:(1) The cultured cells in vitro present typical astrocyte shapes and showed positive GFAP by immunofluorescence were confirmed to be astrocyte.(2) It is no significant effect on cell survival rates under UCNPs-MC540of different concentrations (0μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml)) without laser irradiation (P>0.05)or laser of different energy (0J/cm2,200J/cm2、500J/cm2.1000J/cm2,2000J/cm2) without UCNPs-MC540(P>0.05). when the cells incubated with UCNPs-MC540of various concentrations for12hours underwent laser irradiation of2000J/cm, the cellular survival rates significantly decreased with the increased concentrations (P<0.05). when cells incubated with100μg/ml UCNPs-MC540for12hours underwent laser irradiation of different energy,the cellular survival rates significantly decreased with the increased energy densities (P<0.05).(3) Compared with control, the cells incubated with200μg/ml UCNPs-MC540after photodynamic action show the apoptosis sign, cytochondriome damage, chromatin condensation, cytomembrane crenulation change under observation of transmission electron microscope. Conclusion:(1) Neogenetic SD rats astrocytes were successfully separated, cultured and identificated in vitro.(2)Nanocompound UCNPs-MC540constructed by upconversion fluorescent nanoparticles as carrier of photosensitizer combined with merocyanine540mediated photodynamic therapy have effective killing effect on spinal astrocytes, and may be regarded as a new promising treatment for inhibiting gliosis and glial scar form after spinal cord injury. To a certain extent, Only administrating nanocompound or only effect of light, it is no cell killing and prompt low toxicity of UCNPs-MC540and safe of individual contact with980nm laser.